Erythropoietin is a cytokine required for red blood cell formation. Erythropoietin receptor expression is not restricted to hematopoietic cells and erythropoietin activity extends beyond blood and includes neural protection and cardiac protection demonstrated in select animal models. We found a direct erythropoietin response of coronary artery endothelial cells. Erythropoietin upregulated endothelial nitric oxide synthase activity in vitro and in vivo, and enhanced nitric oxide production. Erythropoietin stimulated signaling pathways, phosphoinositide 3-kinase/protein kinase B and mitogen-activated protein kinase kinase/extracellular signal regulated kinase. Inhibition of phosphoinositide 3-kinase blocked erythropoietin-induced nitric oxide production. To verify the potential of this Epo effect in cardioprotection in vivo, mice with erythropoietin receptor expression in heart restricted to endothelium were treated with erythropoietin. These mice exhibited the cardioprotective erythropoietin response and an increase in endothelial nitric oxide synthase phosphorylation comparable to erythropoietin treated wild type mice. These data suggest that endothelial response and endothelial nitric oxide synthase response in heart is sufficient to provide cardioprotection in this model of ischemic-reperfusion injury in mice.